Synthetic biology has enabled us to artificially convert heterotrophs into autotrophs. Modification of the existing metabolic circuit and induction of enzymes of the Calvin–Benson–Bassham (CBB) cycle including phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) allowed Escherichia coli and Pichia pastoris to fix CO2. This heterotroph to autotroph conversion technology will contribute to reductions in energy consumption and CO2 emissions in the bioindustry.
Heavy-ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse species, including algae, but a preferred mutant cannot be easily obtained without a suitable large-scale screening method. We devised a unique, convenient, and effective method for screening mutants of Haematococcus pluvialis to isolate a strain resistant to environmental stress with low white fluorescence, i.e., a robust strain. Haematococcus was irradiated with heavy-ion beams of carbon ions, argon ions, and iron ions at various doses, after which approximately 10,000 surviving colonies were inoculated into 96-well plates, cultured for approximately 2 weeks, and then left to dry in a refrigerator for 3–12 months without a lid. In these unattended 96-well plates, cells in approximately one-third of the wells died and became white, and the remaining wells were approximately evenly split between red and green. The robustness of wild-type and mutant strains isolated from red and green wells was compared under severe environmental-stress conditions (125 µmol photons m−2 s−1, continuous light period, 45 mM sodium acetate). In the wild-type strain, most cells died, and 93.9% of cells emitted white autofluorescence. In contrast, few G4 cells emitted white autofluorescence, indicating a survival rate of 91.8%. Strains with excellent carotenoid production, such as G7 and R1, showed greater robustness compared to wild-type strains.
In Undaria pinnatifida, an effective method for mutant screening in sporophytes has not been established. The present study developed a novel mutant screening method for Undaria sporophyte by combining gametophyte mutagenesis with heavy-ion beam and land-based tank culture system. When we irradiated gametophytes and sporophytes with carbon- and argon-ion beams, survival rates of the female gametophytes and the sporophytes decreased with increasing dose. However, those of the male gametophyte did not decrease after both of the irradiations. Mutant screening during the sporophyte development was performed by using a land-based tank culture system. High-growth plants were selected in the first mutant (M1) population derived from the irradiated materials. We successfully obtained mutant candidates with higher growth than the wild type in the M2 generation obtained from brother-sister inbreeding of selected M1 plants. Four high-growth mutant candidate lines were selected from M2 populations of 48 lines. The mutant candidates were derived from 3 lines of the gametophyte irradiation and 1 line of the sporophyte irradiation, suggesting that the materials for the irradiation are applicable for mutant induction. The mutant screening method and the selected mutant candidates would advance the breeding and molecular biology in U. pinnatifida.
A mutant showing extra early-flowering and named extra early-flowering4 (exe4) was induced in a previous study by heavy-ion beam mutagenesis of Triticum monococcum strain KU104-1. The exe4 mutant shows heading about 45 days earlier than wild-type KU104-1 in the field. In the present study, we sought to identify the gene that was mutated in exe4 by performing a modified whole-genome sequencing analysis. This analysis exploited a short-read library preparation that uses a modified adaptor and duplex-specific nuclease (DSN) for the efficient elimination of highly repeated sequence elements within genomes. The whole-genome sequence analysis and PCR analysis using an M2 segregation line indicated that the extra-early flowering phenotype of exe4 is associated with a deletion of a gene for a WD repeat protein, named here WHEAT WD REPEAT 1 (WWDR1). Phylogenetic analysis of amino acid sequences showed that the gene is a homolog of Arabidopsis LIGHT-REGULATED WD1 (LWD1) and LWD2, which are circadian clock regulatory genes.
Linear energy transfer (LET) is an important parameter for ion beam mutagenesis. We irradiated rice seeds with carbon ions (LET: 23, 30, 50, or 57 keV µm−1), neon ions (LET: 63 or 70 keV µm−1), or argon ions (LET: 290 keV µm−1). We subsequently isolated 56 mutants and analyzed the mutations in the responsible genes for mutant phenotypes to evaluate the effects of different LETs in rice. Mutations were classified as single nucleotide variants, small deletions (＜100 bp), large deletions (≥100 bp), and chromosomal rearrangements (inversions and translocations). The carbon and neon ions (23–70 keV µm−1) tended to induce small deletions, whereas the argon ions mainly induced large deletions. These results were consistent with the findings of our previous studies on Arabidopsis mutants irradiated with carbon ions (23 or 30 keV µm−1) or argon ions (290 keV µm−1). Moreover, the deletions caused by argon ions were larger than those induced by carbon or neon ions. Therefore, in rice and Arabidopsis, carbon ion (23 or 30 keV µm−1) irradiations are more likely to induce single-gene mutations, whereas irradiation with argon ions (290 keV µm−1) tends to cause large deletions, that can be useful to disrupt tandemly arrayed genes.
Heavy-ion beams have been applied as effective mutagens to various plant materials. Pollen has been used as material for mutant induction and genetic analysis. However, our knowledge of the DNA damage response of plant male gametes remains limited. In the present study, we irradiated Cyrtanthus mackenii pollen with an argon ion beam, which induced complex DNA damage, and investigated the DNA damage response of male gametes during pollen tube growth. Male gametes derived from the irradiated pollen grains were isolated from pollen tubes after 12 and 24 h of culture and subjected to cell cycle analysis. After 12 h of culture, the irradiated generative cells were frequently arrested at metaphase during pollen mitosis II (PMII), and the proportion of metaphase cells increased with increasing absorbed dose. These results suggest that the genomic lesions induced by the argon ion beam caused spindle assembly checkpoint (SAC)-dependent arrest. After 24 h of culture, the irradiated male gametes completed PMII, albeit forming sperm cells with abnormalities in chromosome separation, and chromosomal bridges were often formed between these cells. Moreover, phosphorylated H2AX foci, an indicator of DNA double-strand breaks, were detected in the irradiated male gametes after 24 h of culture, regardless of passing through the SAC. Taken together, these results indicate that male gametes activate functions to cope with radiation-induced complex DNA damage during pollen tube growth.
A high-LET heavy-ion beam has a severe effect on survival and effectively induces chromosomal rearrangements. In this study, the effect of high-LET heavy-ion irradiation on mutation induction in the M1 generation was investigated in an inbred line of Torenia fournieri, which is a widely used horticultural plant. Dry seeds of the inbred line ‘Zairai murasaki’ were irradiated with a C-ion beam (LET: 50 keV µm−1) or Ar-ion beams (LETs: 184 keV µm−1 or 290 keV µm−1) at different doses, and then sown on 1/2 MS plates. After determining the survival rates from each irradiation condition, appropriate doses of each beam were roughly determined to produce a survival rate of 90%: 300, 75, and 50 Gy for the C-ion beam with a LET of 50 keV µm−1, Ar-ion beam with a LET of 184 keV µm−1, and Ar-ion beam with a LET of 290 keV µm−1, respectively. In the screening of branches with aberrant flowers, one and two aberrant plants were isolated from 16 and 30 M1 plants after irradiation with LETs of 184 keV µm−1 and 290 keV µm−1, respectively. However, no aberrant plants were identified in M1 plants after irradiation with a LET of 50 keV µm−1. We concluded that high-LET heavy-ion beam irradiation is effective in inducing mutations even in the M1 generation of inbred ornamental plants. This technique could be widely used for breeding ornamental plants that can be propagated vegetatively.
Cytology requires chromosome specimens, thus, effective preparation methods are needed. Chromosome specimens are frequently prepared from plant root tips. Furthermore, cell cycle synchronization using chemical reagents is applied to obtain a large number of metaphase chromosome specimens. In this study, we focused on the timing of root tip sampling, which is optimal for the preparation of chromosome specimens of the dioecious plant Silene latifolia. The timing was determined from the time seeds were subjected to a germination treatment. Observation of metaphase chromosomes using microscopy revealed that the number of mitotic cells peaked 54 h after the germination treatment. This trend was also observed when the DNA synthesis inhibitor aphidicolin was administered from 24 to 9 h before sampling time points. We used ice-cold treatment for 8, 16, and 32 h as a chromosome condensation method. The 16 h treatment produced suitable chromosome specimens showing satellite ends of chromosomes, whereas the 32 h treatment produced well-condensed chromosome specimens, which were suitable for counting chromosome numbers. Our findings suggest that the timing of root tip sampling is essential for effectively producing plant chromosome specimens.
The evolution of dioecy from hermaphroditism allows for avoidance of self-pollination, and its evolutionary background has been investigated both experimentally and theoretically since it was first proposed by Darwin. To reproduce this evolution, we screened hermaphroditic mutants of Silene latifolia using heavy-ion beam or γ-ray irradiation and characterized the phenotypes of their floral organs. Our scatterplots indicate severe deviations from the trade-off relationships between pollen and ovule numbers and between seed and germinated pollen numbers in hermaphroditic mutant S. latifolia. These deviations presumably led to promotion of dioecy from the ancestral state of S. latifolia. To infer the likely flower phenotypic characteristics of the ancestral plant of S. latifolia before evolving dioecy, the flowers of Silene viscosa, a naturally hermaphroditic plant related to S. latifolia were also characterized. S. viscosa exhibits both spatial separation of stamens from pistils within the flower (reverse herkogamy) and temporal separation of stamen and pistil maturation (dichogamy), raising the question of whether hermaphroditic mutant S. latifolia, which is thought to be the ancestral state, would possess these functions. We show that two hermaphroditic mutants of the dioecious plant S. latifolia exhibit signs of protogyny (reverse dichogamy) and approach herkogamy, as pistils were constantly longer than stamens. These findings illustrate the evolution of dioecy from hermaphroditism as a self-pollination avoidance mechanism and to balance the investments into male and female functions.
Cytological studies of two genera, four species of Commelinaceae from Thailand were conducted. The chromosome numbers and karyotype formulas of the four species analyzed here were Callisia repens, 2n=12, 4sm+8st; Ca. soconuscensis 2n=12, 2sm+10st; Cyanotis axilaris 2n=20, 20st; and Cyanotis cristata 2n=26, 6m+2sm+18st. The karyotypes of the four species were asymmetric, the karyotypes of Cy. axilaris and Cy. cristata have satellite chromosomes. The karyotypes of Cy. axilaris and Cy. cristata are reported for the first time.
In this work, the karyomorphology of eight endemic Thlaspi species (T. cariense, T. violascens, T. densiflorum, T. cataonicum, T. elegans, T. rosulare, T. lilacinum and T. aghricum) has been studied for the first time. The chromosome number is defined to be 2n=2x=12 in T. cataonicum, 2n=2x=14 in T. violascens, T. densiflorum, T. lilacinum, 2n=2x=16 in T. cariense, 2n=2x=18 in T. elegans, T. rosulare, and 2n=2x=20 in T. aghricum. It is concluded that generally, species have symmetrical karyotypes, considering the asymmetry indices. The karyotypes of all the species examined have shown variation. Most of the chromosomes of the studied Thlaspi have centromeres at the median and submedian regions. Systematic and evolutionary perspectives of the studied species are debated as per chromosomal data.
In this study, chromosome length, karyomorphology and chromosome asymmetry of six Turkish Muscari species; Muscari muglaensis, M. nazimiyensis, M. pallens, M. pamiryigidii, M. inundatum and M. fatmaceranie were evaluated. The somatic chromosome number of all examined taxa was determined as 2n=18. The basic chromosome number was x=9. Chromosomes consist of metacentric, submetacentric, and subtelocentric chromosomes. This is the first report on chromosome number and morphology for five of the six taxa studied. Studies on the karyotype and chromosomal diversity of Muscari may provide insight into taxonomic relationships and differences within the genus.
Previous chromosome information is restricted to 13 species in the Asian Begonia sect. Diploclinium. Here we present the first chromosome counts of three species, as well as confirmed their chromosome numbers of five species. Among three species investigated for the first time, the chromosome number of 2n=18 and 2n=32 are new counts in the section. Additionally, we provide the karyotypes of eight species for the first time. Among ca. 135 species in sect. Diploclinium, chromosome numbers of 16 species are available together with the present and previous reports. Among 16 species, they show diverse chromosome numbers with 2n=18, 22, 24, 26, 30, 32, 36, 46, and 54. B. wenshanensis with 2n=18 is the lowest chromosome number in the section and the third lowest chromosome number in Begonia. We suggest that B. coptidimontana with 2n=32 is tetraploid origin with the basic chromosome number of x=8. We discussed the chromosome evolution in Begonia sect. Diploclinium.
The purpose of this experiment is to select Cyclocodon lancifolius (Roxb.) Kurz strain with a high yield and quality to enhance environmental tolerance. Therefore, we report the first case of polyploid induction in C. lancifolius. The treatment of seed and in vitro shoot tips with colchicine was explored as a means of inducing polyploid, and polyploidy was effectively identified by chromosome counting. After successful induction of polyploid, the optimized in vitro culture for polyploid plants was investigated via a complete combination experiment. The results showed that seeds were not suitable for polyploid induction. The shoot tips were treated with 0.05% colchicine solution for 36 h and then transferred to Murashige and Skoog (MS) medium supplemented with 0.1 mg L−1 6-benzylaminopurine (6-BA), 1.0 mg L−1 α-naphthaleneacetic acid (NAA) and 0.05% colchicine for 60 h. Finally, rooting culture was conducted in the above medium without colchicine, yielding the highest induction rate (25.42%), without chimerism. Compared with diploid plants, autotetraploid plants had more distinctive characters, such as larger and darker green leaves, more obvious veins, thicker stems, and more developed root systems. Furthermore, in MS medium supplemented with 1.0 mg·L−1 NAA and 0.01 mg·L−1 6-BA, tetraploid shoots formed complete plantlets through one-step culture, with above 7.0 multiplication coefficient and 100% rooting rate. The survival rate was 100%. The induction system for the autopolyploid of C. lancifolius was successfully established, which provided an experimental basis for breeding heredity and variety improvement.
The genus Cirsium is a member of the tribe Cardueae (Asteraceae) and is distributed in the northern hemisphere. The Carduus-Cirsium group has a long history of taxonomic confusion due to its complicated morphological and molecular phylogenetic data. Here, we report the chromosome numbers and karyotypes of nine currently recognized Korean Cirsium species, as well as their closely related taxa, Carduus crispus, to better understand the chromosomal evolution using classic Feulgen staining. Both diploid (2n=18, 28, 32, and 34) and tetraploid (2n=68) with extensive chromosome number variations including the occurrence of B-chromosomes (0–2Bs), and aneuploidy (2n=30 and 31 instead of 2n=32) have been found in Korean Carduus-Cirsium taxa. The karyotypes of the nine taxa, Cirsium japonicum var. spinissimum, C. schantarense, C. rhinoceros, C. nipponicum, C. setidens, C. vlassoviianum, C. lineare, C. pendulum, and Carduus crispus are reported here for the first time. While these reported chromosome numbers do not provide any delimitation at the sectional levels for the Korean Carduus-Cirsium taxa, karyotypes and chromosome size are well-conserved at the species level and thus, have taxonomic significance.